Enhanced phytase variants

ABSTRACT

The present invention provides the field of enhancing proteins and in particular to that of proteins enhanced by molecular change. It provides a variant of a phytase that is termed enhanced in that is has better thermostability and/or activity than the original phytase. The invention also provides a nucleic acid coding for said variant, a cassette or an expression vector containing said variant, a host cell expressing said variant, a composition comprising said variant and uses thereof, principally in the preparation of food additives and animal feed.

BACKGROUND OF THE INVENTION

The present invention relates to the field of enhancing proteins, inparticular that of proteins enhanced by molecular change. It pertains toa variant of a phytase, termed enhanced, in that it has betterthermostability and/or activity compared with the original phytase. Theinvention also pertains to a nucleic acid coding for said variant, to acassette or an expression vector containing said variant, to a host cellexpressing said variant, to a composition comprising said variant, andalso to the uses thereof, principally in the preparation of foodadditives and animal feed.

Phytate is the principal phosphorus storage compound in plants. Thismolecule, also known as phytic acid or inositol-hexa-phosphate (InsP6 ormyo-inositol hexakisphosphate), consists of a cyclohexane to which sixphosphate groups are bonded. The phytate represents approximately 70% ofplant phosphate, the remaining 30% being present in the free form.Further, phosphate residues of phytate chelate divalent and trivalentcations such as calcium, iron, zinc, magnesium, copper, manganese andmolybdenum, which are essential for nutrition.

Phytases are enzymes that hydrolyze phytate: such enzymes naturallyrelease one or two phosphates, rarely three, which can then be adsorbedinto the digestive system; other reaction products are rarelyinositol-tri-phosphate, principally inositol tetra- and penta-phosphate(FIG. 1). Phytases constitute a family of enzymes that is widelyrepresented in nature: many organisms, from the bacterium to the plantvia fungi and certain animals, express one or more of them. However,mammals do not express any; ruminants or polygastric animals such ascattle and horses have endogenous microorganisms in theirgastrointestinal tract that can degrade the phytate, but this does notapply with monogastric animals, principally pigs and poultry, forexample, and so the phytate originating from ingested plants does notrepresent a useful source of phosphorus. Thus, it is necessary to addfree phosphates to their feed. However, a portion of those added freephosphates and practically all of the phytate ingested by the animalsare discharged into the environment. The phytate is then degraded bybacteria in the ground and ends up in the ground water and rivers in theform of free phosphate. In regions with a high concentration of stockfarming, such agro-industrial phosphate waste, in the free form or inthe form of phytate, represents a major source of pollution, which inparticular results in the proliferation of green algae in rivers andwatercourses. In addition to the esthetic aspect, such algae have amajor ecological impact since they compete with local plant species, inparticular as regards consumption of dissolved oxygen.

One way of being able to use phosphate stored in the form of phytate asa source of phosphate by mammals is to introduce exogenous phytase intofood. Adding said enzyme thus represents an alternative to usinginorganic phosphate as a food supplement. Further, by rendering thephosphate of the phytate accessible, they can also provide betteraccessibility to metallic ions chelated onto the phosphate of thephytate, as well as to proteins bonded to the phytate, rendering themnutritionally available. Phytases are currently used relativelysystematically in animal feed. They are used as a partial replacementfor phosphates and they also render proteins, amino acids, and calciummore accessible.

However, there are several limitations to using phytases in animal feed:

-   -   the insufficient effectiveness of phytases in releasing        phosphates constitutes a first limitation. Phosphates are still        added to animal feed, even in the presence of exogenous        phytases. However, if all of the phytate were to be converted        into free phosphates, supplements would no longer be required;        thus, there is a genuine need to propose more active phytases;    -   the majority of enzymes in current use cannot be added directly        to feed, since they cannot withstand the granulation process,        which involves heating the feed to 95° C. for 90 seconds. The        enzymes are thus sprayed onto the feed after the granulation        stage, which represents an additional cost and constitutes a        second limitation; thus, there is great demand for the        availability of more thermostable phytases.

The aim of the present invention is to overcome current limitations bygenerating a phytase that is sufficiently active to have a substantialimpact on the need for supplementing with phosphates and is sufficientlythermostable to be able to be added directly to the feed of animals,without having to use a spraying technique.

Many phytases have featured in publications and patent applications, orare even already in use in agro-industrial applications. However, noneof them have been able to dispense with the need for supplementing stockfarm animal feed with phosphates, and none of them is sufficientlythermostable to be added directly to animal feed without having to use aspraying technique.

The majority of publications pertain to phytases from Aspergillus nigerand Escherichia coli. Other phytases of microbial origin or derivingfrom plants have also been studied, the problem being to obtainthermostable phytases having a specific activity that is greater thanthat of Aspergillus niger (250 U/mg). The phytases described in theliterature and the patent applications deal with fungi (basidiomycetesand ascomycetes), yeasts and bacteria.

Many phytases have been isolated from fungi and derive in the main fromthe Aspergillus family, but from Absidia, Acrophiaiophora, Agrocybe,Calcarisporiella, Cheatomium, Corynascus, Mucor, Mycelia, Myriococcurn,Penicillium, Peniophora, Rhizomucor, Rhizopus or even from Trametes,Sporotrichum, Neurospora, Trichoderma, Cladosporium, Myceiiophthora,Taleromyces, Thielavia, Humicola, Paxillus and Thermoascus.

A great deal of information is available regarding the specificactivities and Km of such enzymes, such as that in Wyss et al 1999(Appl. Environ. Microbiol. 65 (2) 367-373). Overall, Aspergillusphytases are characterized by high Km values of 5 μM to 20 μM, anoptimum temperature of 57° C. and an optimum pH of 5.5 except forAspergillus fumigatus (opt pH.=6), but have very low specific activitiesranging from 100 to 250 U/mg.

Certain phytases deriving from other fungi have exhibited interestingproperties, in particular high specific activities, such as the phytasefrom Trametes pubescens or the phytase from Peniophora lycii(respectively 1200 U/mg and 1080 U/mg in WO 98/28408). Cladosporium sp.has an interesting phytase with a specific activity of 900 U/mg and a Kmof 15.2 μM, but has a low optimum temperature of 40° C. In addition, the6-phytase from Ceriporia sp. has a specific activity of 11040 U/mg. Somecomplementary information was published by Lassen et al, 2001 (Appl.Environ. Microbiol. 67 (10) 4701-4707, comparing the thermostability ofphytases from basidiomycetes, in particular Peniophora lycii, Ceriporiasp., Trametes pubescens and Aspergillus niger; it appears that saidenzymes have Tms (temperature at which the enzyme is 50% active) thatare quite close, from 55° C. (Trametes pubescens) to 60° C. (Peniophoralycii) but have residual activities (percentage activity resulting frompreincubation for 60 minutes at 80° C. in sodium acetate [0.1 M], pH5.5) that vary widely, from 15% (Trametes pubescens) to 62% (PeniophoraLycii).

Many establishments have filed patent applications concerning saidenzymes, in particular Danisco/Genencor (WO 2001/012792, Penicilliumsubtilis; WO 2003/038035, Trichoderma reesei; WO 2003/038111,Penicillium, Mumicola, Emericella, Fusarium), ABEnzymes/ROAL (EP 0 659215, Aspergillus phytases produced by Trichoderma reesei), DSM/Roche (EP0 684 313, Apergillus terreus, Aspergillus fumigatus, Aspergillusnidulans, Talaromaces thermophilus), BASF (WO 2003/102174, Aspergillus),Adisseo (WO 2003/054199, Penicillium), and Choongang Biotech Ltd. (WO2005/056835, Penicillium oxalicum).

Several bacterial phytases have been described that originate fromBacillus subtilis (Paver and Jagannathan, 1982, Journal of Bacteriology151:1102-1108), Pseudomonas (Cosgrove, 1970, Australian Journal ofBiological Sciences 23:1207-1220), and Klebsiella. Several phytasesoriginating from E. coli have been reported in the literature. Greineret al, in Arch, Biochem. Biophys., 303, 107-113, 1993, purified andcharacterized a novel phytase of E. coli; others have been reported byLim et al., 2000, Nat. Struct. Biol. 7: 108-113, Oshima et al., 1996,DNA Research, 3:137-155, Touati and Danchin, 1987, Biochimie,69:215-221, Rodriguez et al., 2000, Arch. Biochem. Biophys.,382:105-112, Kretz, U.S. Pat. No. 5,876,997 from E. coli B, and appA byDassa et al., 1990, J. Bacteriol. 172:5497-5500.

Mutants from E. coli phytase have been obtained by genetic engineering,resulting in enhanced thermostabilities and specific activities(Rodriguez et al, 2000, Arch. Biochem. Biophys., 382:105-112, Lanahan etal., 2003, US patent application 20030157646). However, none of thosemutations could be used to produce sufficient of those enzymes ofprokaryotic origin in eukaryotic production organisms.

The aim of the present invention is to provide a recombinant enzyme thatis suitable for industrial processes to allow it to be used as a foodadditive, principally in animal feed.

In the application WO 2002/048332, using a BLAST analysis of bacterialgenomes available at the date of the invention and using the appA genefrom E. coli, Diversa identified a novel protein from Yersinia pestishaving phytase activity. That protein had a remarkable feature, namelythat it has a specific activity of 4400 U/mg. No other biochemicalfeatures of that protein were specified in that application. Thatapplication appears to demonstrate a high potential activity for phytaseoriginating from bacteria from the Yersinia family.

On Oct. 20, 2005, the protein sequence with reference ZP_(—)00832361 wasadded to the NCBI database. Said sequence is that of a hypotheticalprotein of Yersinia intermedia ATCC 29909 the corresponding nucleotidesequence for which is presented under reference numeral NZ_AALF01000052,region: 1889 . . . 3214. That protein sequence was obtained bytranslation of the corresponding nucleotide sequence originating fromthe complete sequence for the genome of the strain Yersinia intermediaATCC 29909. Although the PRK10172 domain appears to predict a phytaseactivity, no experimental element accompanied that prediction.

This appears to have been confirmed by the isolation of a very similarphytase from a novel strain of Yersinia intermedia originating from adirt sample from a glacier, denoted H-27 and presented in theapplication WO 2007/128160. The phytase derived from the H-27 strain isdesignated in the NCBI database with accession number AB195370.1 for thenucleotide sequence and DQ986462 for the protein sequence; it has 98%identity as regards the amino acids and 97% identity with the nucleotidesequence encoding the hypothetical phytase of Yersinia intermedia ATCC29909.

The phytase of application WO 2007/128160 has a high specific activityof more than 3000 U/mg, of the same order as the specific activityrecorded for Yersinia pestis in WO 2002/048332. In that application WO2007/128160, the intrinsic biochemical characteristics of the proteinare claimed, namely a molecular weight of 45.5 kDa, an optimum pH in therange 4.0 to 5.0, an optimum temperature in the range 50 to 60° C., atheoretical pI of 7.7, a specific activity of more than 3000 U/mg and ahigh resistance to pepsin and trypsin.

BRIEF SUMMARY OF THE INVENTION

The present invention means that current limits can be exceeded, byproposing an enhanced variant by changing the phytase from Yersiniaintermedia molecularly. The term “enhanced variant” means a variant witha thermostability and/or activity higher than the original phytase fromYersinia intermedia, meaning that it can be used in industrial processesand in particular as a food additive.

As is described in the remainder of the text and with the aid of theaccompanying figures, the present application describes an enhancedvariant of a phytase whose sequence is SEQ ID No. 1 or a functionalderivative thereof, characterized in that it comprises at least onesubstitution on one of the amino acids from the group consisting of P3,V4, A5, P8, T9, G10, V16, V17, L19, S20, R21, H22, G23, V24, R25, S26,P27, T28, K29, Q30, T31, Q32, L33, M34, D36, P39, K41, W45, A49, G50,Y51, L52, T53, G56, A57, V60, Y67, G75, A78, C81, D92, V93, D94, Q95,R96, T97, R98, L99, T100, G101, A103, V116, V125, D126, F129, H130,P131, V132, D133, D140, T142, Q143+H145, A147, L152, P155, L156, E158,E158+S160, F167, A177, C182, G189, D193, N196, F197, K201, K206, P207,T209, K210, V211, S212, L213, L217, A218, L219, S220, S221, T222, L223,G224, E225, I226, F227, L228, L229, Q230, N231, Q233, A234, P236, R242,I250, S251, L252, L253, L255, H256, N257, Q259, F260, D261, M263, A264,Y268, K273, G274, P276, L277, Q292, G293, P297, P300, Q301, G308, G309,H310, D311, T312, N313, I314, A315, N316, G322, A323, Q326, P331, D332,N333, T334, P335, P336, G337, G338, G339, V341, E343, D349, Q352, R353,Y354, I355, A370, E371, K376, P379, A380, G381, D388, E391, S393, G394and P414, the positions being indicated in SEQ ID No.1. Preferably, theenhanced phytase variant whose sequence is SEQ ID No.1 or a functionalderivative thereof comprises at least one substitution selected from thegroup consisting of P3L, P3V, V4G, ASP, P8N, P8V, T9I, T9Q, T9S, T9Y,G10A, G10P, V16M, V17W, L19G, S20C, R21F, H22A, H22S, H22Y, G235, V24C,R25C, S26C, P27F, T28N, T285, T28V, K29N, Q30C, Q30D, Q30R, Q32R, L33R,M34C, D36N, P39N, K41G, W45C, G50D, G50E, Y51G, Y51N, Y51Q, Y51W, L52C,L52G, T53C, G56C, A57C, V60I, Y67F, Y67W, G75R, A78P, C81N, D92R, V93G,D94G, D94S, Q95N, Q95V, R96A, T97N, R98N, R98T, L99C, G101C, A103C,V116C, V125N, D126Q, F129W, H130N, H130Q, H130R, H130W, H130Y, P131S,V132W, D133G, D133P, D133R, D133V, D133W, D140E, D140F, D140N, T142N,Q143N+H145T, A147C, L152N, L152P, P155N, P155T, L156N, E158N,E158N+5160T, F167N, A177N, A177S, A177T, C182N, G189N, D193C, N196C,F197V, K201N, K206A, P207N, P207S, P207T, T209C, K210C, K210E, K210N,K210S, K210T, K210V, K210Y, V211C, V211G, S212N, L217N, A218N, L219V,S220N, L223S, E225D, F227S, L229H, Q230N, Q230T, N231K, Q233S, Q233T,A234K, P236N, R242N, I250S, I250T, S251N, L252M, L255T, H256A, H256E,H256P, N257I, Q259K, Q259S, Q259T, Q259Y, D261F, M263L, A264N, A264P,Y268C, Y268E, Y268N, K273N, G274C, G274S, P276L, Q292P, G293N, P297N,P297S, P300N, Q301L, G308S, H310N, H310R, D311A, D311E, D311G, T312D,T312N, T312P, T312V, N313F, N313R, I314E, I314M, N316C, G322C, A323E,Q326S, Q326T, P331R, D332A, D332L, D332N, D332Q, N333V, P335C, P335G,P335R, P335S, P335T, G339C, V341A, V341E, E343A, E343G, D349N, D349S,D349T, Q352S, Q352T, R353C, Y354N, I355W, A370D, A370T, E371S, E371T,K376S, P379L, P379S, P379T, A380S, A380T, G381S, D388C, E391N, S393G,G394S, G394T and P414Q, the positions being indicated in SEQ ID No.1.Preferably, the enhanced phytase variant whose sequence is SEQ ID No.1or a functional derivative thereof comprises at least one combination ofsubstitutions selected from the substitutions of the preceding group.

SEQ ID No.1 is the sequence appearing in the NCBI database filed on Oct.20, 2005 with accession number ZP_(—)00832361, but not, however,containing the 23 amino acid signal sequence at the 5 end of theprotein. SEQ ID No.1 thus corresponds to residues 24-441 appearing underthe above-mentioned accession number. SEQ ID No.1 also contains thenucleic acid sequence coding for the preceding protein sequence in theNCBI base with reference NZ_AALF01000052. A nucleic acid coding for avariant of the present invention can readily be prepared on the basis ofthis sequence using techniques that are well known to the skilledperson, for example by directed mutagenesis of the codon to be modified,to obtain the desired amino acid substitution. Thus, the sequence forthe enhanced phytase variant of the present invention corresponds to SEQID No.1 including the selected substitution or substitutions.

SEQ ID No.2 reproduces only the protein sequence of SEQ ID No.1.

In a particular embodiment, the enhanced variant of the presentinvention comprises a single substitution.

In a preferred embodiment, the enhanced variant of the present inventionor a functional derivative thereof comprises at least one substitutionon one of the amino acids from the group consisting of K29, Q30, Y51,L52, G75, C81, V93, Q95, R98, L99, F129, H130, D140, T142, P155, F167,A177, G189, K201, K210, L219, I250, S251, L252, L255, M263, Y268, G274,Q292, G293, P297, G308, N316, Q326, D349 and E391, the positions beingindicated in SEQ ID No.1. In another preferred embodiment, the enhancedvariant of the present invention or a functional derivative thereofcomprises substitutions on one of the amino acids from the groupconsisting of K29, Q30, Y51, L52, G75, G81, V93, Q95, R98, L99, F129,H130, D140, T142, P155, F167, A177, G189, K201, K210, L219, I250, S251,L252, L255, M263, Y268, G274, Q292, G293, P297, G308, N316, Q326, D349and E391, the positions being indicated in SEQ ID No.1. Preferably, thesubstitutions on the amino acids K29, Q30, Y51, L52, G75, C81, V93, Q95,R98, L99, F129, H130, D140, T142, P155, F167, A177, G189, K201, K210,L219, I250, S251, L252, L255, M263, Y268, G274, Q292, G293, P297, G308,N316, Q326, D349 and E391, are selected from the group consisting ofK29N, Q30D, Y51G, Y51N, Y51Q, Y51W, L52G, G75R, C81N, V93G, Q95N, R98T,L99C, F129W, H130Y, D140F, D140N, T142N, P155N, P155T, F167N, A177N,A177S, A177T, G189N, K201N, K210N, K210S, L219V, 12505, 1250T, S251N,L252M, L255T, M263L, Y268N, G274C, Q292P, G293N, P297N, G308S, N316C,Q326S, Q326T, D349S, D349T and E391N, the positions being indicated inSEQ ID No.1.

In a yet more preferred embodiment, the enhanced variant of the presentinvention or a functional derivative thereof comprises substitutions onone of the amino acids from the group consisting of K29, Q30, Y51, L52,G75, V93, R98, L99, F129, H130, D140, T142, P155, F167, A177, K201,K210, L219, 5251, L252, L255, M263, Y268, G274, Q292, G293, G308, N316,Q326 and E391, the positions being indicated in SEQ ID No.1. Preferably,the substitutions on the above amino acids are selected from the groupconsisting of K29N, Q30D, Y51G, Y51Q, Y51W, L52G, G75R, V93G, R98T,L99C, F129W, H130Y, D140F, T142N, P155T, F167N, A177N, A177S, A177T,K201N, K210S, L219V, S251N, L252M, L255T, M263L, Y268N, G274C, Q292P,G293N, G308S, N316C, Q326S, Q326T and E391N, the positions beingindicated in SEQ ID No.1.

In another particular embodiment, the enhanced variant of the presentinvention or a functional derivative thereof comprises a combination ofsubstitutions selected from the group consisting of G274C+N316C,T142N+A177T+Q326T, K210S+Y268E+Q292P, D140F+Y268E+Q292P,F167N+Y268E+Q292P, T142N+A177T+K210S+Q326T,T142N+A177T+K210S+Y268E+Q292P+Q326T,T142N+A177T+K210S+Y268E+Q292P+Q326T+G274C+N316C,L52C+L99C+T142N+A177T+K210S+Y268E+Q292P+Q326T, the positions beingindicated in SEQ ID No.1. In a preferred mode of this particularembodiment, the enhanced variant of the present invention or afunctional derivative thereof comprises a combination of substitutionsconsisting of T142N+A177T+K210S+Y268E+Q292P+Q326T, the positions beingindicated in SEQ ID No.1. In another particularly preferred mode of thisembodiment, the enhanced variant of the present invention or afunctional derivative thereof comprises a combination of substitutionsconsisting of T142N+A177T+K210S+Y268E+G274C+Q292P+N316C+Q326T, thepositions being indicated in SEQ ID No.1. In another preferred mode ofthis particular embodiment, the enhanced variant of the presentinvention or a functional derivative thereof comprises a combination ofsubstitutions consisting of T142N+A177T+K210S+Q326T, the positions beingindicated in SEQ ID No.1. In another preferred mode of this particularembodiment, the enhanced variant of the present invention or afunctional derivative thereof comprises a combination of substitutionsconsisting of G274C+N316C, the positions being indicated in SEQ ID No.1.In another preferred mode of this particular embodiment, the enhancedvariant of the present invention or a functional derivative thereofcomprises a combination of substitutions consisting of T142N A177TQ326T, the positions being indicated in SEQ ID No.1. In anotherpreferred mode of this particular embodiment, the enhanced variant ofthe present invention or a functional derivative thereof comprises acombination of substitutions consisting of K2105 Y268E Q292P, thepositions being indicated in SEQ ID No.1. In another preferred mode ofthis particular embodiment, the enhanced variant of the presentinvention or a functional derivative thereof comprises a combination ofsubstitutions consisting of D140F+Y268E Q292P. In another preferred modeof this particular embodiment, the enhanced variant of the presentinvention or a functional derivative thereof comprises a combination ofsubstitutions consisting of F167N+Y268E+Q2921).

The present invention provides an enhanced variant of a phytase whosesequence is SEQ ID No.1 or a functional derivative thereof comprisingthe selected substitution or substitutions.

The present invention also provides a nucleic acid coding for anenhanced phytase variant in accordance with the present invention or afunctional derivative thereof, an expression cassette comprising anucleic acid of the present invention, and a vector comprising a nucleicacid or an expression cassette of the present invention. The vector maypreferably be selected from a plasmid, a phage, a phagemid and a viralvector.

The present invention also provides a composition comprising at leastone enhanced phytase variant the sequence for which is SEQ ID No.1 or afunctional derivative thereof with the selected substitution orsubstitutions in accordance with the present invention. It also providesany solid, liquid or gaseous mixture comprising a certain percentage ofat least one enhanced phytase variant of the present invention. It alsoprovides mixtures preferably containing one, two, three, four, five orten enhanced phytase variants in accordance with the present inventionor functional derivatives thereof. The present invention also providesphytase preparations or compositions containing a certain percentage ofat least one enhanced phytase variant of the present invention or afunctional derivative thereof and one or more other enzymes havingadvantageous properties.

The present invention provides the use of an enhanced phytase variant ofthe invention or a functional derivative thereof, for the preparation ofa food additive. Using an enhanced phytase variant of the presentinvention or a functional derivative thereof is of concern to industrialprocesses that can be used to liberate minerals and in particularphosphate from plants, either in vitro when treating food beforeingestion using the enhanced phytase variant of the present invention,or in vivo by administering said variant directly to animals before orwith their feed.

The present invention provides the use of a nucleic acid, an expressioncassette or a coding vector and/or containing at least one enhancedphytase variant whose sequence is SEQ ID No.1 or a functional derivativethereof with the selected substitution or substitutions of the presentinvention, to transform or transfect a host cell. It also provides ahost cell comprising a nucleic acid, an expression cassette or a vectorcoding for an enhanced phytase variant of the present invention or afunctional derivative thereof. The present invention also provides theuse of said nucleic acid, said expression cassette, said vector or saidhost cell to produce an enhanced phytase variant of the presentinvention or a functional derivative thereof. It also provides a methodof the production of an enhanced phytase variant of the presentinvention, comprising transforming or transfecting a host cell with anucleic acid, an expression cassette or a vector of the presentinvention, culturing the transformed or transfected host cell andharvesting the enhanced phytase variant or a functional derivativethereof produced by the host cell. The host cell may be prokaryotic oreukaryotic. Thus, the host cell may be a microorganism, preferably abacterium, a yeast or a fungus. The host cell may also be a mammaliancell such as a COS7 or CHO cell.

The term “functional derivative” means any enzyme derived from thephytase variant of the present invention comprising structuralmodifications while retaining phytase activity. Such modifications may,for example, involve extending the enzyme by adding new domains, orpartial or complete substitutions of domains such as replacing stretchesof amino acids by amino acids from other enzymes that might provideother functions/properties. The term “functional derivative” alsoincludes a dimerized form of the variant of the enzyme of the presentinvention, which may be homo- or heterodimeric, or even polymeric,having enhanced properties such as thermostability, for example, becauseof domain multiplication. The term “functional derivative” alsoencompasses a chimeric form of the phytase variant of the presentinvention, fused with another protein/enzyme of interest or with one ormore domains of said enzyme of interest. The term “functionalderivative” also encompasses a functional fragment of the phytasevariant of the present invention that preserves phytase activity. Saidactivity may be measured using one of the protocols described inExamples 4 and 5. The fragment may comprise 250, 275, 300, 325, 350,375, 380, 385, 390, 395, 400, 405, 410 or 415 consecutive amino acids ofthe phytase of the present invention. Said functional fragment may alsobe dimerized or polymerized and/or fused with another protein/enzyme ofinterest or with one or more domains thereof.

The term “variant” or “mutant” means a nucleotide sequence havingmutations compared with a reference nucleotide sequence. Said mutationsmay be silent due to degeneracy of the genetic code; the protein encodedby the variant is then identical to the protein encoded by the referencenucleotide sequence. Said mutations may also cause substitutions ofamino acids in the protein encoded by the variant compared with theprotein encoded by the reference nucleotide sequence. The term “variant”includes sequences containing mutations obtained by directedmutagenesis. The expression “variant” is attributed to nucleotidesequences as well as to protein sequences encoded. by said nucleotidesequences, presenting said mutations.

The enhanced phytase variant of the present invention or a functionalderivative thereof may comprise substitutions on one of the amino acidsfrom the group consisting of P3, V4, A5, P8, T9, G10, V16, V17, L19,S20, R21, H22, G23, V24, R25, S26, P27, T28, K29, Q30, T31, Q32, L33,M34, D36, P39, K41, W45, A49, G50, Y51, L52, T53, G56, A57, V60, Y67,G75, A78, C81, D92, V93, D94, Q95, R96, T97, R98, L99, T100, G101, A103,V116, V125, D126, F129, H130, P131, V132, D133, D140, T142, Q143+H145,A147, L152, P155, L156, E158, E158+S160, F167, A177, C182, G189, D193,N196, F197, K201, K206, P207, T209, K210, V211, S212, L213, L217, A218,L219, S220, S221, T222, L223, G224, E225, I226, F227, L228, L229, Q230,N231, Q233, A234, P236, R242, I250, S251, L252, L253, L255, H256, N257,Q259, F260, D261, M263, A264, Y268, K273, G274, P276, L277, Q292, G293,P297, P300, Q301, G308, G309, H310, D311, T312, N313, I314, A315, N316,G322, A323, Q326, P331, D332, N333, T334, P335, P336, G337, G338, G339,V341, E343, D349, Q352, R353, Y354, I355, A370, E371, K376, P379, A380,G381, D388, E391, S393, G394 and P414 not described in the group P3L,P3V, V4G, ASP, P8N, P8V, T91, T9Q, T9S, T9Y, G10A, G10P, V16M, V17W,L19G, S20C, R21F, H22A, H22S, H22Y, G23S, V24C, R25C, S26C, P27F, T28N,T28S, T28V, K29N, Q30C, Q30D, Q30R, Q32R, L33R, M34C, D36N, P39N, K41G,W45C, G50D, G50E, Y51G, Y51N, Y51Q, Y51W, L52C, L52G, T53C, G56C, A57C,V60I, Y67F, Y67W, G75R, A78P, C81N, D92R, V93G, D94G, D94S, Q95N, Q95V,R96A, T97N, R98N, R98T, L99C, G101C, A103C, V116C, V125N, D126Q, F129W,H130N, H130Q, H130R, H130W, H130Y, P131S, V132W, D133G, D133P, D133R,D133V, D133W, D140E, D140E, D140N, T142N, Q143N+H145T, A147C, L152N,L152P, P155N, P155T, L156N, E158N, E158N+5160T, F167N, A177N, A177S,A177T, C182N, G189N, D193C, N196C, F197V, K201N, K206A, P207N, P207S,P207T, T209C, K210C, K210E, K210N, K210S, K210T, K210V, K210Y, V211C,V211G, S212N, L217N, A218N, L219V, S220N, L223S, E225D, F227S, L229H,Q230N, Q230T, N231K, Q233S, Q233T, A234K, P236N, R242N, I2505, 1250T,S251N, L252M, L255T, H256A, H256E, H256P, N257I, Q259K, Q259S, Q259T,Q259Y, D261F, M263L, A264N, A264P, Y268C, Y268E, Y268N, K273N, G274C,G274S, P276L, Q292P, G293N, P297N, P297S, P300N, Q301L, G308S, H310N,H310R, D311A, D311E, D311G, T312D, T312N, T312P, T312V, N313F, N313R,I314E, I314M, N316C, G322C, A323E, Q326S, Q326T, P331R, D332A, D332L,D332N, D332Q, N333V, P335C, P335G, P335R, P335S, P335T, G339C, V341A,V341E, E343A, E343G, D349N, D349S, D349T, Q352S, Q352T, R353C, Y354N,I355W, A370D, A370T, E371S, E371T, K376S, P379L, P379S, P379T, A380S,A380T, G381S, D388C, E391N, S393G, G394S, G394T and P414Q, the positionsbeing indicated in SEQ ID No.1, or combinations of substitutions derivedfrom said group, as mentioned above. As an example, said substitutionsmay be substitutions termed “conservative”, i.e. substitutions within agroup of amino acids having similar or equivalent characteristics, suchas amino acids with low steric hindrance, or acidic, basic, polar,hydrophobic and aromatic amino acids in accordance with the table below:

Low steric Ala (A) Gly (G) Ser (B) Thr (T) hindrance Acid Asp (D) Glu(H) Basic Arg (R) His (H) Lys (K) Polar Asn (N) Gln (Q) Hydrophobic Ile(I) Leu (L) Met (M) Val (V) Aromatic Phe (F) Tyr (Y) Trp (W)

Thus, for example, the enhanced variant of the present invention or afunctional derivative thereof may comprise substitutions equivalent tothe substitution P276L described in the previous group, such as thesubstitutions P276I, P276M or P276V using the classification in theabove table. The above interpretation also applies to combinations ofsubstitutions, further, the enhanced phytase variant of the presentinvention or a functional derivative thereof may comprise othermutations that are not described in this group, preferablysubstitutions, in particular some that are known in the field. In aparticular embodiment, the enhanced phytase variant or a functionalderivative thereof comprises a maximum of 40, 35, 30, 25, 20, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 substitutions or 1 substitutionrelative to the wild type phytase, in particular relative to SEQ IDNo.1.

The term “enhanced variant” means a variant having enhanced properties,in particular thermostability and/or specific activity and/or enhancedexpression, relative to the parent phytase. In addition, the enhancedvariant of the present invention may have greater resistance toproteolysis by proteases or others. The enhancement to one of moreproperties of the enhanced phytase variant of the present invention isat least 5%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100%, or by a factor of 2, 5, 10 or 100 compared with theproperties of the parent phytase, measured under the same experimentalconditions. In a preferred embodiment, said enhancements amount to atleast 20%. The thermostability of the phytase may be measured usingprocedures that are detailed in Example 5. The specific activity of thephytase may be measured using the procedures detailed in Example 4.Phytase expression may be measured using the procedures detailed inExamples 2 and 3.

Visualizing model structures in 3D using software such as swiss-model(http://www.expasy.ch/) and spdbv v4.01 (GlaxoSmithKline) often meansthat hypothetical explanations can be constructed by comparison withstructural modifications to enzymes that cause changes in activityand/or properties, particularly as regards the possible bonds betweenadjacent amino acids. When taking a predictive approach, suchvisualizations also mean that certain residues can be targeted formutagenesis experiments. As an example, when enhanced thermostability isdesired, the targeted residues may be those that can stiffen thesecondary structure. Said stiffening may be accomplished in differentmanners; as an example, the targeted residues may be substituted with aproline residue that conventionally generates fewer rotamers and thusstiffens the secondary structure to which it belongs. In addition,stiffening of the secondary structures may be accomplished by generatingnew hydrogen bonds and new saline bridges; visualizing model structuresin 3D means that residues that can establish such bonds withstructurally close residues can be targeted. When enhancedthermostability and/or activity is desired, an approach other than thevisualization of 3D models is to modify the charges carried by a residueand the ensuing steric stresses. It is known that in some circumstancesthe substrate/product of an enzyme participates in stabilizing the 3Dconformation of the enzyme and may provide increased thermostability. Itis clear that visualizing such model structures in 3D means thatresidues can be focused upon for enhancing other parameters of theenzyme of industrial interest such as activity, expression or resistanceto proteolysis, for example. It is also clear that this approach byvisualizing model structures in 3D is a predictive tool allowingmutagenesis strategies to be constructed without in any way guaranteeingany enhancement in enzymatic properties.

The term “expression vector” means that the expression vector may be anytype of recombinant vector (in particular a plasmid, virus, etc),enabling the nucleotide sequence of the enhanced variant of the presentinvention to be expressed. The choice of this expression vector dependson its compatibility with the targeted expression host in which it istransformed or transfected. Said vector may be linear or a closedcircle. It may replicate autonomously, i.e. it may be anextrachromosomal entity replication of which is independent of thechromosome of the host containing it, a plasmid, an extrachromosomalelement, a mini-chromosome or an artificial chromosome. In contrast,when it is introduced into the host cell, the vector may be integratedinto the genome of the host for replication at the same time thereof.Equally, several vectors may be necessary for expression of the enhancedvariant of the present invention and may be used simultaneously, as wellas a transposon.

The vectors allowing expression of the enhanced variant of the presentinvention may contain one or more markers that allow easy selection oftransformed or transfected host cells. Said selection markers aretypically genes the product of which provides their host with anadvantage and, for example, produces bacterial resistance to anantibiotic, prototrophy for auxotrophs, resistance to heavy metals, etc.Examples of bacterial selection markers are genes that provideresistance to antibiotics such as ampicillin, kanamycin, tetracyclin andchloramphenicol in particular. Particular examples of markers suitablefor selection in yeasts are the genes ADE2, HIS3, LEU2, LYS2, MET3, TRP1and URA3. Particular examples of markers used in filamentous fungi areamdS (acetamidase), argE (ornithin carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidin-5′-phosphate decarboxylase), sC (adenyltransferase sulfate)and trpC (anthranilate synthase), in particular. The vectors allowingexpression of the enhanced variant of the present invention do not haveto contain selection markers.

With autonomous replication, the vector must contain an origin ofreplication adapted to the host cell. Particular examples of bacterialorigins of replication are those of the plasmids pBR322, pUC19, pACYC177and pACYC184 for replication in Escherichia coli, and pUB110, pE194,pTA1060 and pAM[beta] for replication in Bacillus. Non-exhaustiveexamples of origins of replication in yeasts are the 2-micrometerorigins of replication ARS1, ARS4, the combination of ARS1 and CEN3 andthe combination of ARS4 and CENG. The origin of replication may alsocontain a mutation that means that it can be sensitive to temperature.Examples of origins of replication for use in filamentous fungi are AMA1and ANSI from Aspergillus nidulans (Gems et al., 1991, Gene 98:61-67;Cullen et al., 1987, Nucleic Acids Research 15: 9163-75; WO 00/24883).

With integration into the genome of the host cell, the vector must allowits integration by means of the coding sequence for the enhanced variantof the present invention or any other suitable sequence in the vector,via homologous or non-homologous recombination. it may also containadditional nucleic acid sequences to direct its integration into thegenome of the host cell. in order to maximize the chances of integrationinto the genome of the host, the integration sequences must be ofsufficient length, such as 100 to 10000 base pairs, preferably 400 to10000, more preferably 800 to 10000 base pairs. The integrationsequences may be coding or non-coding.

The 23 codon “signal” nucleotide sequence present at the 5′ end of thegene for Yersinia intermedia phytase (denoted in the NCBI by numbersNZ_AALF01000052 and ZP_(—)00832361) and cleaved in the mature formcontributes to secretion of the enzyme in its host of origin. Thepresence of such a sequence is conventional and well known to theskilled person. Changing this sequence and replacing it with a suitablesequence is also a tactic that is well known to the skilled person whenexpression of the gene under consideration is desired in anotherorganism or in another cellular compartment via a plasmidic vector orother vector selected to suit the desired organism for expression. Thus,said sequence may be replaced by the signal sequence for other genessuch as that for PelB, PhoA, OmpA or β-lactamase in particular, for itsexpression in a prokaryotic host. During expression in a yeast such asPichia pastoris, the signal sequences present at the 5 end of the genefor the phytase of Yersinia intermedia may be the signal sequences PHO1and αfactor respectively from the genes for a phosphatase and theαfactor of that organism. During expression in Saccharomyces cerevisiae,the same αfactor signal sequence may be used. During expression inYarrowia lypolytica, the signal sequence present at the 5′ end of thegene for the phytase from Yersinia intermedia may be the XPR2 signalsequence from the same XPR2 gene of a protease of said organism.

The term “host cell” means that the cell may be prokaryotic oreukaryotic; it may be a gram positive bacterium such as Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, Bacillus thuringiensis,Streptomyces lividans or Streptomyces murinusque in particular, or agram negative bacterium such as Escherichia coli or Pseudomonas sp., forexample; this list is not limiting. The present invention also providesa method of the production of a phytase or a variant thereof that issoluble and active in a bacterium, preferably Escherichia coli,comprising expression of a nucleic acid encoding the phytase or avariant thereof in a bacterium, preferably Escherichia coli, andoptionally recovering the phytase so expressed.

The host cell may be a yeast from the genus Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia,in particular. The host cell may preferably be Saccharomycescarisbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, Saccharomyces oviformis, Kluyveromyces lactis, Pichiapastoris or Yarrowia lipolytica.

The host cell may be a filamentous fungus from the genus Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Mvceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma. The host cell may preferably be Aspergillusawamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Caldariomyces fumago, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulfureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta,Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinuscinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa,Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenurn, Phanerochaete chrysosporium, Phlebia radiata, Pleurotuseryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor,Trichoderma harzianum, Trichoderma koriingii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride.

The host cell may be a mammalian cell such as COS7 or CHO (U.S. Pat. No.4,889,803; U.S. Pat. No. 5,047,335).

The term “nucleic acid” means DNA (cDNA or gDNA), RNA or a mixture ofthe two. The nucleic acid may comprise modified nucleotides comprising,for example, a modified bond, a modified puric or pyrimidic base, or amodified sugar. It may be prepared using any method known to the skilledperson, including chemical synthesis, recombination, mutagenesis, etc.

The nucleic acid coding for an enhanced phytase variant of the presentinvention may be optimized in terms of the codons constituting it tomaximize its expression in a particular host that differs from itsorganism of origin. Since the universal genetic code is degenerate,several codons (codon=triplet of nucleotides) exist that code for agiven amino acid. These homonymic codons are not used randomly, sincethe corresponding tRNAs (transfer RNA) do not exist in all cells in thesame concentrations. This means that certain codons then have lesschance of being expressed in tissues where the corresponding tRNA israre. This fact, which is well known to the skilled person, is animportant parameter to be considered when carrying out expression in agiven host that differs from the organism of origin of a particulartransgene. Tables of the frequency of use of codons in a particularorganism have been published and are well known to the skilled person.Thus, the nucleic acids coding for the enhanced phytase variants of thepresent invention may have to be optimized in order to promote theirexpression in a selected production host.

The term “percentage identity” or “identity” between two nucleic acid oramino acid sequences in the context of the present invention means apercentage of nucleotides or amino acid residues that is identicalbetween the two sequences to be compared, obtained after the bestalignment, said percentage being purely statistical and the differencesbetween the two sequences being distributed randomly over their entirelength. The best alignment or optimum alignment is the alignment forwhich the percentage identity between the two sequences to be compared,as calculated below, is the highest. Comparisons of sequences betweentwo nucleic acid or amino acid sequences are traditionally carried outby comparing these sequences after having aligned them in an optimizedmanner, said comparison being carried out in comparison segments orwindows to identify and compare local regions with sequence similarity.As well as carrying it out manually, sequences may be optimally alignedfor comparison using the Smith and Waterman (1981). local homologyalgorithm (Ad. App. Math. 2: 482), using the Neddleman and Wunsch localhomology algorithm (1970) (J. Mol. Biol. 48: 443), using the Pearson andLipman similarity search method (1988) (Proc. Natl. Acad. Sci. USA 85:2444), or employing software programs using those algorithms (GAP,BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.). The percentageidentity between two nucleic acid or amino acid sequences is determinedby comparing these two aligned sequences in an optimized manner by meansof a comparison window in which the region of the nucleic acid or aminoacid sequence to be compared may include additions or deletions comparedwith the reference sequence for optimized alignment between those twosequences. The percentage identity is calculated by determining thenumber of identical positions for which the nucleotide or the amino acidresidue is identical for the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 to obtain thepercentage identity between those two sequences. Thus, amino acids thatare conserved or equivalent to those present in the enhanced phytasevariant of the present invention may be discerned in phytases from otherorganisms. Thus, the enhancement provided by the selected substitutionsin the enhanced phytase variant of the present invention may bediscerned by carrying out an equivalent substitution in a phytase for anorganism other than that from which the phytase of the present inventionoriginates. Clearly, such equivalent substitutions fall within thepurview of the present invention.

The enhanced phytase variant of the present invention may be used in thereactions and methods mentioned above in a purified or partiallypurified form. Purification of the enhanced phytase variant of thepresent invention may be basic and in particular carried out by lysisand filtration of the contents of flasks or production containers and/orby centrifuging steps, and/or by successive selective precipitationsusing ammonium sulfate and/or by evaporation. Said basic procedures canbe used to obtain fractions of the enhanced phytase variant of theinvention exhibiting a large increase in specific activity. Purificationof the enhanced variant of the invention may be complete and requirevarious steps that are well known to the skilled person, in particularchromatography (ion exchange, affinity, hydrophobic, size exclusion), orelectrophoresis (preparative, by isoelectric concentration) [ProteinPurification, J. C. Janson and Lars Ryden, VCH Publishers, New York,1989].

The purified or partially purified fraction of the enhanced phytasevariant of the present invention may be used in the reactions andmethods mentioned above, in the immobilized or non-immobilized form.Methods of immobilizing the enhanced variant of the present invention onorganic or inorganic supports are well known to the skilled person.These supports may in particular be polyacrylamides, agaroses,celluloses, sephadexes or dextrans, porous glass beads, or aluminum ortitanium hydroxides.

The term “composition” generally means a composition comprising at leastone enhanced phytase variant whose sequence is SEQ ID No.1 with theselected substitution or substitutions of the present invention. It alsoprovides any solid, liquid or gaseous mixture comprising a certainpercentage of at least one enhanced phytase variant of the presentinvention. It also provides mixtures containing one, two, three, four,five or ten enhanced phytase variants of the present invention.

In general, the compositions containing the phytases are liquid orso-called “dry” compositions.

Liquid compositions do not have to contain anything other than highlypurified phytase. However, stabilizers such as glycerol, sorbitol ormonopropylene glycol may be added. Said composition may also containother additives such as salts, sugars, preservatives, pH buffers,proteins and phytate. Typically, the liquid compositions are aqueouscompositions or oil-based suspensions. The liquid compositions may beadded to the food before or after an optional granulation step.

So-called “dry” compositions may be compositions that are dried byfreezing, spraying, or they may be extruded dry compositions; under suchcircumstances, said composition does not have to contain anything otherthan the enzyme in its dry form. The dry compositions may also begranules that can be mixed or are ready to be mixed with a foodcomponent, or to form a pre-mix component. The size of the enzymegranules is preferably compatible with that of the other components ofthe mixture. This represents a safe and practical way of incorporatingone or more enzymes into the food.

As an example, a stable enzyme formulation may be prepared by spraying aliquid phytase mixture onto a component such as soya meal then dryingthe assembly. The reduction in the moisture content and bindinginteractions of the phytase with the component protect the enzyme fromexternal environmental factors such as the extreme temperatures employedduring manufacture of the food component. In addition, presenting thephytase preparation in the dry form may improve its stability byreducing the activity of potential proteolytic enzymes that may bepresent in trace amounts at the end of the liquid fermentation stepsduring the production method. The dry phytase preparation may, forexample, be used as a food supplement in the poultry and pig productionindustry.

Starting from a dry enzyme preparation, granules are prepared usingagglomeration techniques that are well known to the skilled person, in amixer in which a filler material and the enzyme are co-agglomerated toform granules. The granules are prepared from matrices onto which theenzyme may be absorbed or onto which a layer of enzymes may be applied.Typical materials that can serve as a matrix are salts such as disodiumsulfate. Other potential matrices may be based on talc, clay, magnesiumsilicate, aluminum silicate or cellulose fibers. Optionally, bindingagents such as dextrins may be included in the granules.

Entraining agents may be included, in any form of the followingcomponents: starch, manioc, potato, rice, wheat, corn etc. Salts mayalso be added.

Optionally, the granules may be coated with specifically dedicatedmixtures, in particular hydrophobic mixtures, based on palm nut oil,beef suet and, if necessary, other additives such as calcium carbonateor clay.

Further, the phytase preparation may contain other agents such ascolorants, aromatic compounds, stabilizers, vitamins, minerals as wellas other enzymes or mixtures of enzymes having advantageous properties.This is particularly true for pre-mixes.

The term “food additive” means a component that is practically pure or acomposition containing several components intended to be added to afood. In particular, said additive is intended to become a fully-fledgedcomponent of said food and is intended to affect, modify or enhance oneor more properties of said food. Thus, a phytase preparation used as afood additive means a phytase that is not a natural component of thefood to which it is added or that is not present in that food in itsnatural concentration, or that is added separately from the othercomponents of the food, alone or in association with other foodadditives. Typically, a food additive contains several components suchas vitamins, minerals, entraining agents, excipients, other enzymes ormixtures of enzymes with advantageous properties.

The term “phytase preparation as a food additive ready for use” or“phytase as a ready to use food additive” means a food additive that isnot produced in situ in the food or the animal feed. Such a phytase orpreparation may be given directly as a food to humans or to animals,preferably directly after mixing with the other constituents of saidfood. As an example, a food additive in accordance with this aspect ofthe present invention is combined with other compounds in order toproduce a food. These other compounds include one or more other enzymes,preferably thermostable, vitamin-containing food additives, mineral foodadditives, or amino acids as food additives. The result of this mixtureor this combination of compounds may be mixed, in appropriateproportions, with other components such as protein or cereal supplementsto form the final food. The methods of manufacturing said food may becarried out using any apparatus that is well known to the skilledperson, such as a double granulation machine, a steam granulator, anexpander or an extruder.

The term “phytase preparation” or “phytase composition” as used in thepresent invention means preparations of compositions that contain asignificant quantity of at least one enhanced phytase variant of thepresent invention and one or more other enzymes having advantageousproperties for the preparation of food. Such enzymes may appear on thefollowing non-exhaustive list: alpha-galactosidases,beta-galactosidases, in particular lactases, other phytases,beta-glucanases, in particular endo-beta-1,4-glucanases andendo-beta-1,3(4)-glucanases, cellulases, xylosidases, galactanases, inparticular arabinogalactan-endo-1,4-beta-galactosidases andarabinogalactan-endo-1,3-beta-galactosidases, endoglucanases, inparticular endo-1,2-beta-glucanase, endo-1,3-alpha-glucanase, andendo-1,3-beta-glucanase, enzymes that degrade pectins, in particularpectinases, pectinesterases, pectin lyases, polygalacturonases,arabinanases, rhamnogalacturonases, rhamnogalacturonan-acetyl-esterases,rhamnogalacturonan-alpha-rhamnosidase, pectate lyases,alpha-galacturonisidases, mannanases, beta-mannosidases,mannan-acetyl-esterases, xylan-acetyl-esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phospholipasesand cutinases.

Supplementation of the animal feed additive in accordance with thepresent invention may be carried out before or simultaneously with ameal. Preferably, supplementation is carried out at the same time as themeal.

An effective quantity of phytase that may be added to food isapproximately 10 PPU/kg to 20000 PPU/kg of food; preferably in the range10 PPU/kg to 15000 PPU/kg; more preferably in the range 10 PPU/kg to10000 PPU/kg, in particular 100 PPU/kg to 5000 PPU/kg, particularly 100PPU/kg to 2000 PPU/kg of food.

The scope of the invention also includes the use of an enhanced phytasevariant of the present invention in the manufacture of foods intendedfor human or animal consumption. Grain or flour intended for human foodmay be treated with the phytase to reduce their phytate content,allowing an increase in the nutritional value of said products byincreasing the availability of essential minerals such as iron, calciumand zinc, for example. Beyond the nutritional value, such a treatmentwith phytase may enhance the efficiency of production of that food. Asan example, adding phytase to white soya flakes during the soya proteinextraction process may enhance the yield and quality of the extractedproteins. The phytase is active during the manufacture of the food, butnot in the final product. This is particularly true when producing andbaking dough for baked items. Similarly, in the production of animalfeed, soya or rapeseed grain may be pre-treated with phytase beforetheir final manufacture and/or conditioning. Such pre-treatment meansthat anti-nutritional elements such as phytate can be degraded and thequality of the nutritional value of the food in question can beenhanced. The phytase may then optionally still be active in thedigestive tract of the animals after ingesting the food.

The scope of the invention also encompasses the use of an enhancedphytase variant of the present invention as an agent facilitating foodtransformation. In particular, the phytase of the present invention maybe used as a supplement in human food to facilitate digestion. As anexample, one or more tablets containing a suitable quantity of phytasemay be ingested by an individual before eating in order to provide thatindividual's digestive tract with an active enzyme. The benefit ofingesting phytase is particularly remarkable when eating food thatcannot be treated with the phytase during its manufacture.

The phytase of the present invention may advantageously be used withmono- or polygastric animals, in particular young cattle. Diets intendedfor fish and crustaceans may also be treated with the phytase in orderto improve the conversion yields between the food supplied and growth ofthe animals, and also to reduce the quantities of phosphate excreted inintensive production systems. The food treated in accordance with thepresent invention may be provided to poultry (turkeys, ducks, geese,partridge, hens, broilers), to pigs, horses, cattle, sheep and goats,dogs or cats. is of particular application to poultry and pigs,including but not being limited to hens, broilers, turkeys, ducks andgeese.

The phytase of the present invention is used to produce novelcombinations of food ingredients or food with advantageous qualities. Asan example, it may be used to produce food with a reduced inorganicphosphate content. This quantity is adjusted as a function of thequantity and activity of the added phytase present in the final food, oractive in one of the food ingredients forming part of the composition ofthe final food. Preferably, such a food can contain ingredients such asmicro-nutrients, vitamins, amino acids and effective and optimizedquantities of phytase and inorganic phosphate such that the quantity ofphytase is in the range 50 to 20000 units of phytase per kilo of foodand the quantity of inorganic phosphate is less than 0.45%. Preferably,these two quantities are in the range 100 to 10000 units of phytase perkilo of food and less than 0.225% of inorganic phosphate; morepreferably in the range 150 to 10000 units of phytase per kilo of foodand less than 0.15% of inorganic phosphate; still more preferably in therange 250 to 20000 units of phytase per kilo of food and with no addedphosphate. These novel combinations are of broad interest, such as inreducing phosphate discharges into the environment and optimizing theconversion yields between the food supplied and animal growth, which isparticularly sought-after in intensive stock farming.

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS

The present invention is described below in more detail in the followingexamples, which are in no way limiting in nature, with the aid of theaccompanying figures and tables:

Table 1: Mutants isolated using the THR™ approach;

Table 2: List of mutants with an additional glycosylation siteclassified as a function of their percentage accessibility to therespective solvents;

Table 3: List of pairs of mutations allowing the addition of additionaldisulfide bridges;

Table 4: 80%-20% activity extinction coefficients various mutants:

Table 4A: 80%-20% activity extinction coefficients for the mutantsK210S, Y268E and Q292P;

Table 4B: Details of data for calculating the 80%-20% activityextinction coefficient of the mutant K210S;

Table 4C: 80%-20% activity extinction coefficients for the mutantsT142N, A177T and Q326T;

Table 4D: 80%-20% activity extinction coefficients for the G274C/N316Cmutant;

Table 5: Lists of mutants targeting enhanced activity:

Table 5A: List of positions targeted by a distance of 10 Angstrom orless about the catalytic enzyme site;

Table 5B: List of substitutions targeting an enhancement to the activitycharacterized in a first series of experiments;

FIG. 1: Degradation of phytate by a phytase;

FIG. 2: Principle of THR™ technique;

FIG. 3: Measurement of residual activities of the mutants PHY-98-4X andPHY-98-6X produced by Saccharomyces cerevisiae after pre-heating for 0to 2 minutes at 80° C.;

FIG. 4: Measurement of residual activities of the mutants PHY-98-4X andPHY-98-6X produced by Saccharomyces cerevisiae after pre-heating for 15minutes at temperatures of 45° C. to 65° C.;

FIG. 5: Measurement of residual activities of the mutants PHY-98-4X andPHY-98-6X produced by Saccharomyces cerevisiae after pre-heating for 1minute at temperatures of 60° C. to 80° C.;

FIG. 6A: Measurement of residual activities of the mutants PHY-98-6X andPHY-98-6X-ss11 produced by Pichia pastoris after pre-heating for 0 to 30minutes at 80° C.;

FIG. 6B: Measurement of residual activities of the mutants PHY-98-6X andPHY-98-6X-ss11 produced by Pichia pastoris after pre-heating for 0 to 5minutes at 80° C.;

FIG. 7: Measurement of the relative activities of the mutants PHY-98-4Xand PHY-98-6X, compared with the enzyme of origin PHY-98, produced bySaccharomyces cerevisiae, as a function of time;

FIG. 8: 12% SDS-PAGE gel of production supernatants for various mutantsexpressed by Pichia pastoris, digested or not digested by theendoglycosidase Hf.

DETAILED DESCRIPTION OF THE INVENTION Examples Example 1 ObtainingEnhanced Phytase Variants from Yersinia intermedia Plasmidic Constructs:

Producing enhanced phytase variants of the present invention requiredthe construction of various plasmidic vectors that were capable ofcarrying out the directed mutagenesis experiments necessary in order toobtain libraries of variants or mutants as well as for the expression ofsaid mutants in various screening or production hosts.

Constructs in pET25b:

-   -   Using molecular biological techniques that are well known to the        skilled person, the ORF (Open Reading Frame) ZP_(—)00832361        corresponding to the sequence NCBI ATCC 29909 and to the        corresponding nucleotide sequence NZ_AALF01000052, region: 1889        . . . 3214 was cloned into the plasmidic vector pET25b. Before        cloning, the original signal sequence (first 23 amino acids) was        deleted from the ORF and replaced by the signal sequence for the        phytase of Escherichia coli. This signal sequence for the        phytase of Escherichia coli had been cloned into the vector        pET25b in order to optimize expression of the phytase from        Yersinia intermedia in E. coli. The vector pET25b could also be        used to express the protein of interest in the form of a fusion        protein with a “6H is tag” facilitating isolation and/or        purification of the protein of interest using methods that are        well known to the skilled person. This vector containing the        phytase from Yersinia intermedia fused with the signal sequence        of the phytase from E. coli was transformed in a BL21(DE3) E.        coli strain from which the AppA gene coding for the endogenous        phytase of E. coli had been deleted.

Constructs in pNCK:

-   -   The ORF of ZP_(—)00832361 was cloned into the vector pNCK using        molecular biological techniques that are well known to the        skilled person. This vector can be used to express, in a        thermophilic microorganism Thermus thermophilus, a fusion        protein between the protein of interest and a thermostable        kanamycin resistance gene using the method described in patent        application WO 2006/134240 and corresponding to Biométhodes'        THR™ technique.

Constructs in pYES2:

The ORF of ZP_(—)00832361 was cloned into the vector pYES2 usingmolecular biological techniques that are well known to the skilledperson. This vector, containing the signal peptide for the phytase ofAspergillus niger in the 5′ position of the ORF ZP_(—)00832361, allowedexpression of the phytase from Yersinia intermedia by Saccharomycescerevisiae.

Constructs in pPIC9:

-   -   The ORF of ZP_(—)00832361 was cloned into the vector pPIC9 using        molecular biological techniques that are well known to the        skilled person. This vector, containing the signal peptide of        the αfactor of Pichia pastoris (Invitrogen) in the 5′ position        of the ORF ZP_(—)00832361, allowed expression. of the phytase        from Yersinia intermedia by Pichia pastoris.        Construction of Libraries of Mutants in pNCK, Screening and        Obtaining Enhanced Variants:

Several libraries of mutants of the phytase from Yersinia intermediawere created using Biométhodes' Massive Mutagenesis® technique describedin U.S. Pat. No. 7,202,086 or in Saboulard et Si (Biotechniques, 2005Sep 39(3): 363-8). Briefly, mutagenic oligonucleotides were synthesizedto produce phytase mutants on each of the amino acids constituting theenzyme. The libraries of mutants were constructed from the vector pNCKcontaining the gene of the phytase using the Massive Mutagenesis®protocol described in in U.S. Pat. No. 7,202,086 or in Saboulard et al(Biotechniques, 2005 Sep 39(3): 363-8), then transformed and amplifiedin the Escherichia coli strain DH10B. The mutants of the phytasecontained in these libraries were then screened using the THR™ techniquedescribed in patent application WO 2006/134240, the principle of whichis summarized in FIG. 2. The possibility offered by the system forselecting, in a single step, a very large number of mutant moleculesmeans that highly diverse libraries can be worked with. Such a library,targeting all of the residues of the protein, approximately 10⁸ clones,was constructed using Massive Mutagenesis®.

Briefly, the libraries of mutants were transformed in cultures ofThermus thermophilus that had been rendered competent. The transformantswere set to grow in liquid medium at 70° C. to produce pre-cultures thatwere then spread onto a solid medium containing stringent concentrationsof kanamycin of the order of 25 μG/mL. After incubating for 48 hours at70° C., only the mutants with a protein structure that resisted theselection temperature and that were folded correctly could allowfunctional folding of the thermostable kanamycin-resistant gene and thuscould grow in the presence of kanamycin. Using this approach, variousmutants were isolated, and their plasmidic DNA was isolated andsequenced. The various mutants revealed a total of 138 substitutions inthe 89 positions shown in Table 1. The various substitutes wereintroduced individually into the phytase cloned into the plasmidicvector pET25b in order to be expressed in Escherichia coli andcharacterized in more detail as regards their activity and theirthermostability. Example 4 details the protocols for measuring theactivity used and Example 5 details the protocols for characterizing theresidual activity of the mutants as a function of temperature. A firstseries of experiments was able to identify at least 5 mutants with anenhanced thermostability relative to the wild type enzyme. Of these, 3mutants respectively contained substitutions on the amino acids K210,Y268 and Q292, particularly the substitutions K210S, Y268E and Q292P.These mutants had an 80%-20% residual activity extinction coefficient ofrespectively 1.75, 1.98 and 2.30 as can be seen in Table 4A. Theseindices were calculated by the ratio between the differences intemperatures allowing firstly 80% of residual activity to be retainedand secondly 20% of residual activity to be maintained for the variousvariants compared with the wild type enzyme. Details of the calculationof this index are shown for the mutant K210S in Table 4B.

Visualizing model structures in 3D using software such as theswiss-model (http://www.expasy.ch/) and spdbv v4.01 (GlaxoSmithKline)means that certain explanations can be advanced concerning the increasesin thermostability obtained for the 3 mutants mentioned above. K210 is apotentially solvent-accessible residue located in a secondary“sheet”-like structure, possibly involved in bonding and/or interactionwith the substrate (phytate) or the products derived from the reaction.K210 thus seems to be an important residue in particular because of itspositive charge and its large volume, which could generate steric andelectrostatic constraints as regards access of the substrate to oregress of products from the active site. The loss of this positivecharge in the K210S substitution and modification of the associatedspatial constraint could thus in general perturb the reaction (access ofsubstrate+egress of products+solvation of the active site cavity) andmodify the kinetic constants thereof, more particularly the apparent Kmsfor the various substrates generated during the reaction. In somecircumstances, it is known that the substrates/product could participatein stabilizing the 3D structure of the protein and provide increasedthermostability. It might be envisaged that this substitution could, forexample, facilitate positioning of the substrate by increasing thenumber of conformers tolerated in the active site and/or could alsofacilitate evacuation of the phosphates liberated during the reaction,preventing them from being too numerous in the cavity of the activesite, which presence could clearly perturb the enzyme-substrate complexand destabilize the whole structure. Y268 is a potentiallysolvent-accessible residue located in the secondary “loop” typestructure. By modeling the Y268E substitution using the softwarementioned above, it can be established that the number of potentialhydrogen bonds with structurally close residues (with the CO peptidefunction of the Q143 residue for example) are increased, but also thatthis substitution could produce an additional saline bridge, inparticular with K146. Overall, the region in question becomes stiffer,which could explain the observed gain in thermostability. Q292 is also apotentially solvent-accessible residue located in a secondary “loop”type structure. Substitution by a proline residue is a relativelyconventional engineering approach in this type of secondary structurewith poor conservation. This type of residue generates few rotamers, andas a consequence stiffens the secondary structure in which it is locatedand thus can sometimes result in increased thermostability that couldthus bring about the substitution Q292P.

Construction of Mutants with Additional Glycosylation Sites

Certain post-translational modifications such as glycosylations havebeen described as being protein stabilizers. The signals forN-glycosylations in a protein sequence are NxT or NxS. Such sites wereintroduced into the phytase of Yersinia intermedia cloned into thevector pYES2 either using the protected Biométhodes Massive Mutagenesis®technique mentioned above or using a mutagenesis technique that is wellknown to the skilled person, such as overlapping PCR, by introducing a Tresidue at the +2 position relative to a residue N present in thephytase of Yersinia intermedia or by introducing a residue N at the −2position relative to an S or T residue. The positions into which theglycosylation sites were introduced are classified as a function oftheir percentage accessibility to solvents (% ASA) using the softwareavailable at:http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py?from=ASA (T.J. Richmond, Solvent accessible surface area and excluded volume inproteins. J. Mol. Biol, 178, 63-89 (1984). Preferably, 22 substitutionson residues with a percentage accessibility to solvents of >35% wereselected. More preferably, 10 substitutions on residues with apercentage accessibility to solvents of >70% were selected. Thesedifferent variant constructs allowing the addition of glycosylationsites are summarized in Table 2 as a function of their respectivepercentage accessibility to solvents.

The mutant constructs were transformed in Saccharomyces cerevisiae andcharacterized in more detail as regards their activity andthermostability. Example 5 details the protocols for characterizing theresidual activity of the mutants as a function of temperature. A firstseries of experiments was able to identify several mutants with anadditional glycosylation site having enhanced thermostability comparedwith the wild type enzyme. These mutants contain substitutions on theamino acids T142, A177 and Q326 characterized by a percentageaccessibility to solvents of >70%. More particularly, the substitutionson said amino acids are T142N, A177T and Q326T. These mutants have arespective 80%-20% residual activity extinction coefficient of 1.62,1.62 and 1.52, as shown in Table 4C. These indices were calculated fromthe ratio between the differences in temperatures allowing firstly 80%of the residual activity and secondly 20% of the residual activity to beretained for the various variants compared with the wild type enzyme.Details of the calculations for this index are shown in Table 4B for themutant K210S.

Construction of Mutants Having Additional Disulfide Bridges

Several pairs of residues were identified as regards their distance andorientation compatible with the formation of a disulfide bridge andreplaced with cysteine residues. These pairs were identified byvisualizing model or homologous structures of phytases in pdb formatusing a Swisspdb Viewer (GlaxoSmithKline) type program, taking intoaccount optimized distances between residues and orientation of the sidechains thereof. Using this approach, 12 pairs of residues located atapproximately 2 Angstrom were selected and are listed in Table 3.

The above residues were introduced into the phytase from Yersiniaintermedia cloned into the vector pYES2, either using the protectedBiométhodes Massive Mutagenesis® technique as mentioned above or using amutagenesis technique that is well known to the skilled person such asoverlapping PCR. The mutant constructs were transformed in Saccharomycescerevisiae and characterized in more detail as regards their activityand thermostability. Example 5 details the protocols for characterizingthe residual activity of mutants as a function of temperature. A firstseries of experiments was able to identify one mutant with an additionaldisulfide bridge site and enhanced thermostability. This mutant containstwo substitutions on the residues G274 and N316, more particularly thesubstitutions G274C and N316C. This mutant had an 80%-20% residualactivity extinction coefficient of 2.33, as shown in Table 4D. Thisindex was calculated by the ratio between the differences intemperatures allowing firstly 80% of the residual activity and secondly20% of the residual activity to be retained for the variant SS11compared with the wild type enzyme. Details of the calculations for thisindex for the mutant K210S are shown in Table 4B.

Construction of Mutants Targeting Enhanced Activity:

Several residues were targeted in order to identify mutants having anenhanced activity. These residues were identified by visualizing modelor homologous structures of phytases in the pdb format using a SwisspdbViewer (GlaxoSmithKline) type program. The criterion for selection. thatwas selected was: residues located at a distance of 10 Angstrom or lessaround the catalytic site of the enzyme. Using this approach, 76positions were selected and are listed in Table 5A. Complete diversityby using the oligonucleotides NNS was introduced at these positions intothe phytase of Yersinia intermedia cloned into the pYES2 vector, eitherusing the protected Biométhodes Massive Mutagenesis® technique asmentioned above or using a mutagenesis technique that is well known tothe skilled person, such as overlapping PCR. Thus, for each of thepositions listed in Table 5A, the mutants individually comprising one ofthe 19 possible substitutions from the list of 20 existing amino acidswere constructed; using the standard single letter code, these 20 aminoacids are: A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y andV. The mutant constructs were transformed in Saccharomyces cerevisiaeand characterized in more detail as regards their respective activityand thermostability. Example 5 details the protocols for characterizingthe residual activity of the mutants as a function of temperature. Afirst series of experiments allowed 14 mutants containing thesubstitutions listed in Table 5B to be characterized.

Combination of Enhancements:

The various approaches employed meant that the thermostability and/oractivity and/or proteolysis resistance gains could be accumulated byscreening and characterizing the various mutants in each of theapproaches: THR™, adding glycosylation sites, adding disulfide bridgesand targeting activity. A first series of experiments was used toconstruct several mutants combining the thermostability enhancementsobtained by screening libraries using THR™, adding new glycosylationsites and adding disulfide bridges. These mutants were constructed usingthe protected Biométhodes Massive Mutagenesis® technique mentionedabove. Several mutants were constructed comprising combinations ofsubstitutions: G274C+N316C, T142N+A177T+Q326T, K210S+Y268E+Q292P,D140F+Y268E+Q292P, F167N+Y268E+Q292P, T142N+A177T+K210S+Q326T,T142N+A177T+K210S+Y268E+Q292P+Q326T,T142N+A177T+K210S+Y268E+Q292P+Q326T+G274C+N316C. The mutant constructswere transformed in Saccharomyces cerevisiae and characterized in moredetail as regards their activity and their thermostability. Example 5details the protocols for characterizing the residual activity ofmutants as a function of temperature. FIGS. 3 to 7 show the results ofcharacterizations of some of these mutants, in particular the mutantsPHY-98-4X, PHY-98-6X and PHY-98-6X-SS11 respectively containingcombinations of mutations T142N+A177T+K210S+Q326T,T142N+A177T+K210S+Y268E+Q292P+Q326T andT142N+A177T+K210S+Y268E+Q292P+Q326T+G274C+N316C, the positions beingindicated in SEQ ID No.1. Three bacterial strains respectivelycontaining the enzyme of origin PHY-98 and the mutants PHY-98-6X andPHY-98-6X-SS11 mentioned above, cloned into the plasmidic vector pYES2as described above, form the subject matter of a deposit of biologicalmaterial under respective references CNCM I-4172, CNCM I-4173 and CNCMI-4174, on Jun. 24, 2009. These various mutants may act as a basis foradding one or more additional substitutions deriving from variousselective approaches and show one/more enhancements inactivity/thermostability in order to create novel combinations ofmutations accumulating the enhancements or demonstrating synergisticeffects in the enhancements due to these novel combinations ofmutations.

Example 2 Expression of Enhanced Variants of the Phytase from Yersiniaintermedia in Saccharomyces cerevisiae

The plasmid pYES2 containing the enhanced phytase variant from Yersiniaintermedia as described above was transformed by electroporation in theyeast strain Saccharomyces cerevisiae ΔPho4 and the transformants wereselected on SD-U solid medium. Several clones were pre-cultured inliquid SD-U medium overnight at 30° C., with agitation. Saidpre-cultures allowed more culture to be seeded in 2% YP Galactoseproduction medium. They were produced overnight at 30° C., withagitation.

Example 3 Expression of Enhanced Variants of the Phytase from Yersiniaintermedia in Pichia Pastoris

The plasmid pPIC9 containing the enhanced phytase variant from Yersiniaintermedia as described above was transformed in cells of Pichiapastoris that had been rendered competent. After selecting coloniescontaining the plasmid, one colony was grown for 16 hours at 28° C.,with agitation, in 50 mL of BMG medium to form a pre-culture. Theproduction of variants was controlled by using different induction timesin 0.5% methanol depending on the desired quantity of said variant.Before induction, the optical density (OD) of the pre-cultures wasmeasured at 600 nm; for induction, optimized ODs of 2 to 60D units wererequired. The pre-cultures were then centrifuged and re-suspended in BMMmedium containing 0.5% methanol at an initial OD in the range 1 to 30ODu/mL depending on the envisaged induction time: 1 ODu/ml, for 96 hoursof induction, 6 ODu/mL for an induction of 72 hours and 30 ODu/ml, foran induction of 48 hours. 100% methanol was added every 24 hours to givea final concentration of 0.5%. These production procedures were employedfor Erlenmeyer flask production procedures adapted to volumes of 10 mLto 200 mL.

For higher volumes, production was carried out in a 5 L to 50 Lbioreactor adapted to production volumes of 2 L to 20 L. The type ofbioreactor used (Applikon) could automatically monitor yeast growth bycarrying out regular measurements of the optical density at 600 nm andthe oxygen pressure (pO₂), optimizing maintenance of the inductionconditions with methanol. The procedure used was adapted from theprotocol for fermentation in Pichia pastoris from Invitrogen (“Pichiaexpression kit; a manual of methods of expression of recombinantproteins in Pichia pastoris” • Version H dated 11 Jan. 2002). FIG. 8shows a 12% SDS-PAGE gel of production supernatants for various mutantsexpressed by Pichia pastoris, digested or otherwise by theendoglycosidase Hf (New England Biolabs P0703). The gel tracks denotedPhy 98, Phy 98 6x and Phy 98 6x SS11 correspond to 10 μG of productionsupernatants from the molecules PHY-98, PHY-98-6X and PHY-98-6X-SS11described in Example 1. The mutant Phy 98-6X-SS5 is a mutant containingthe substitutions L52C+L99C+T142N+A177T K2105 Y268E+Q292P+Q326T. ThePhy98 tracks show the migration profile for wild type phytase ofYersinia intermedia, not glycosylated, since it does not have thepotential N-glycosylation sites NxS/T; this is illustrated by theabsence of differences in migration after digestion with theendoglycosidase Hf. The tracks Phy 98 6x, Phy 98 6x SS5 and Phy 98 6xSS11, in the absence of digestion by the endoglycosidase Hf, show themigration profiles of mutant phytases with high molecular masses due torespective glycosylation of the mutants. This glycosylation is shown,after digestion with the endoglycosidase Hf (tracks denoted “digestedEndo Hf”), by a return of the migration profile for the various mutantstowards that of the wild type phytase even if digestion by theendoglycosidase may be incomplete in some circumstances.

Example 4 Measurement of the Activity of Enhanced Phytase Variants fromYersinia intermedia

The activity of variants of the phytase from Yersinia intermedia as wellas the activity of the protein of origin used as the control weremeasured using a colorimetric test measuring the phosphate liberated inthe presence of a solution of phytate used as the substrate. In brief,40 μL of supernatant from the test sample (diluted or not in a sodiumacetate buffer) or 40 μL of a calibrating solution of a phosphate weremixed with 40 μL of phytate (20 g/L). The reaction was incubatedconventionally for 15 minutes at 37° C. The reaction was stopped with 80μL of 0.5 M NaOH or 20% TCA. 60 μL of the total volume of the reactionof 160 μL was transferred to be revealed with 60 μL of a Fe—Mo solution.The revealing reaction was left for 15 minutes in the dark before beingread in a spectrophotometer at 620 nm. All of the reaction solutionswere produced using WFI phosphate-free water. The reaction buffer was asodium acetate buffer, 0.25 M, pH 4.5. The substrate used was a phytatesolution produced in the above reaction buffer from a 200 g/L stocksolution. The revealing solution was formed extemporaneously using 4volumes of Mo solution mixed with 1 volume of Fe solution. The Mosolution was a solution of 0.012 M molybdate and the Fe solution was a0.38 M iron II solution.

In order to calculate the activity, a phosphate calibration curve wasproduced with a KH₂PO₄ range of 0 to 10 μmol/mL. By using the reactionconditions described above, the phytase activity of the test samples wascalculated by applying the following formula: phytase activity inU/mL=[(OD 620 nm)×(dilution factor)]/[(slope of calibrationcurve)×(reaction time in minutes)].

The protein concentrations were calculated using the conventionalBradford method familiar to the skilled person.

FIG. 7 shows that the relative activities as a function of time (0 to 15minutes) were higher for the mutants PHY-98-4X and PHY-98-6X comparedwith the enzyme of origin PHY-98 in Saccharomyces cerevisiae.

Example 5 Characterization of Thermostability of Enhanced PhytaseVariants of Yersinia intermedia

The thermostability of variants isolated from the phytase of Yersiniaintermedia was determined by measuring a residual activity of varioussupernatants from production of the variants either after pre-heating toa constant temperature for varying times or after a fixed pre-heatingtime at varying temperatures. In the first alternative, the variantswere pre-heated to 80° C. for times of 0 to 30 minutes. In the secondalternative, two types of residual variant activity were measured eitherafter pre-heating for 15 minutes to temperatures of 45° C. to 65° C., orafter pre-heating for 1 minute to temperatures of 60° C. to 80° C. Theresidual activities were determined in the first alternative as the %activity of the same sample without pre-heating or in the secondalternative as the % of the activity of the same sample pre-heated tothe first measured temperature, the two reference activities beingconsidered as the 100% points. The residual activity is shown as rawvalues for the optical density at 600 nm after pre-heating for 15minutes in FIG. 4.

FIGS. 3, 4 and 5 show the results obtained for the variants PHY-98-4Xand PHY-98-6X expressed by Saccharomyces cerevisiae compared with theenzyme of origin PHY-98. FIG. 3 shows the higher residual activities ofthe two mutants PHY-98-4X and PHY-98-6X after pre-heating for 0 to 2minutes at 80° C. compared with the enzyme of origin PHY-98. FIG. 4shows the high residual activities of the two mutants PHY-98-4X andPHY-98-6X after pre-heating for 15 minutes at temperatures varying from45° C. to 65° C. FIG. 5 shows the high residual activities of the twomutants PHY-98-4X and PHY-98-6X beyond 60° C. using 1 minute ofpre-heating.

FIGS. 6 a) and 6 b) show the high residual activities of the mutantsPHY-98-6X and PHY-98-6X-SS11, expressed by Pichia pastoris, afterpre-heating times at 80° C. of respectively 0 to 30 minutes and 0 to 5minutes.

Example 6 Thermostability of Mutants in Granulation Tests

Granulation tests could be carried out to determine the thermostabilityof the various mutants relative to the wild type and existing and/orcommercially available enzymes. The various phytases could beincorporated into methods of forming and formulating granules intendedto be added to animal feed, for example.

These granules could be formed by mixing/kneading supernatants from theproduction of the mutants and reference enzymes that have to becompared, for example a matrix composed of corn starch and water, underthe same conditions. The granulation matrix may contain differentrelative phytase/corn/water percentages. Conventionally, after kneading,the matrix can be extruded with an extruder similar to the NICA™ E-220type and spheronized directly using a NICA or Fuji Paudal™ QJ-400G typespheronizer. The particles obtained are then dried in a Glatt GPCG 1.1type fluidized bed drier. The phytase activity in the granules isgenerally in the range 2500 to 3000 FTU/g.

The granules formed may be mixed with food. Depending on the volume ofthe tests, the quantity of food formed may vary. As an example, 250 g ofgranules may be mixed with 25 kg of food to form a pre-mix. Just beforethe test, this pre-mix may be incorporated into 225 kg of food, forexample, with the same composition. In non-limiting manner, a typicalpoultry feed may be composed of 45% to 50% corn, 0 to 5% peas, 0 to 4.5%rape flour, 0 to 4.5% sunflower seed flour, 0 to 2.5% corn flour gluten,6% to 10% whole soya beans, approximately 25% soya meal, approximately4% tapioca, 1% to 3.5% of soya oil, 0 to 4% of animal fat, 0.5% to 1% ofa cocktail of vitamins (Mervit 100), approximately 1% of powdered chalk,0.2% to 1.3% of monocalcium phosphate, 0.1% to 0.4% of salts, 0 to 0.3%of sodium bicarbonate (NaHCO₃), 0.05% to 0.3% of L-lysine, 0.15% to0.25% of DL-methionine and 0 to 0.05% of L-threonine. A pre-mix ofapproximately 25 kg can typically be mixed in a Collete MP90 typeplanetary mixer for 10 minutes. A mixture of the order of 225 kg can bemixed in a Nauta type 1200 liter mixer. Samples of this mixture aretaken at this stage to determine the activity and stability of themutants and the reference phytases before forming the final granules. Amixture of the order of 250 kg is typically dosed into themixer/conditioner using a dosing screw at a rate of approximately 600kg/hour where it is heated by injecting steam at approximately 95° C.The total residence time is approximately 10 to 30 seconds, after whichthe hot mixture is directed towards a granulating press. For the tests,the sizes of the granules that could be produced were of the type 5/45mm (width/length) or 3/65 mm. The temperature of the granules at theoutlet from the press is typically 82° C. to 83° C. for the first typeof granules and 91° C. to 93° C. for the second type. Following thisstep, the granules are cooled on a cooling mat from which samples aretaken in order to determine the activity and stability of the mutantsand reference phytases after formation of the final granules.Granulation yields in terms of activity may thus be obtained for eachmutant, compared with the reference enzymes, by producing activityreports after and before the granulation step. A protocol for measuringphytase activity is given in Example 4. In addition, a standard protocolfor measuring phytase activity adapted to these methods has beenpublished with the following reference: van Engelen et al., Journal ofAOAC International 1994, 77:760-764.

Example 7 Tests in Animal Feed Trials

Several approaches could be used to measure the effectiveness of themutants of the invention in liberating phosphate from phytate in vivo inorder to contribute to animal growth compared with reference phytases.

Various animals such as pigs could be integrated into well-establishedprotocols. These had free access to water and a typical dietconstituted, for example, by 67% corn, 28% soya flour, 1% powderedchalk, 0.1% L-lysine, 1% corn oil, 0.25% of a conventional vitamincocktail, 0.5% salts, 0.5% antibiotics. The feed waste was collecteddaily. The weight gain of the animals was measured weekly to calculatethe mean gain per day, the mean daily food intake and the gain/intakeratio. The mutants with a particular advantage and the best performanceswere typically those which had an increased gain/intake ratio.

In addition, in vitro models exist that can simulate digestion in thetract of a monogastric animal. As an example, feed samples composed of30% soya flour and 70% corn flour may be supplemented with calciumphosphate in an amount of 5 g/kg of feed and preincubated at 40° C., pH3 for 30 minutes, followed by adding pepsin in an amount of 3000 U/g offeed and various dosages of phytase in the range 0 (blank control) to 1U of phytase/g of feed. Various phytase mutants could be tested andcompared with reference phytases. The various samples were incubated at40° C., initially at a pH of 3 for 60 minutes then at a pH of 4 for 30minutes. The reactions were then stopped and the phytate and inositolphosphates were extracted by adding hydrochloric acid in a finalconcentration of 0.5M, incubating for 2 hours at 40° C., followed by afreeze-thaw cycle and one hour's incubation at 40° C.

The phytate and the inositol phosphates were separated by highperformance ion exchange chromatography as described by Q. C. Chen, andB. W. Li (2003), Journal of Chromatography A 1018, 41-52 as well as byE. Skoglund, N. G. Carlson, and A. S. Sandberg (1997), J. Agric. FoodChem. 45, 431-436. The phosphate that was liberated was calculated fromthe difference between the phosphate bound to the inositol phosphates inthe samples treated with phytase compared with the samples not treatedwith a phytase. The mutants of interest released a larger quantity ofphosphate.

Biological Material Deposits:

Three bacterial strains containing the constructs PHY-98, PHY-98-6X andPHY-98-6X-SS11 used in the above examples were deposited with theCollection Nationale de Cultures de Microorganismes (CNCM) at theInstitut Pasteur, 25, Rue du flocteur Roux, 75724 Paris Cedex France,under the terms of the Treaty of Budapest:

Identifying Accession numbers references of Deposit for biologicaldeposited strains date material received PHY-98 Jun. 24, CNCM I-41722009 PHY-98-6X Jun. 24, CNCM I-4173 2009 PHY-98-6X-SS11 Jun. 24, CNCMI-4174 2009

LITERATURE CITED

-   D. J. Cosgrove (1970) inositol phosphate phosphatases of    microbiological origin, inositol phosphate intermediates in the    dephosphorylation of the hexaphosphates of myo-inositol,    scyllo-inositol, and D-chiro-inositol by a bacterial (Pseudomonas    sp.) phytase. Australian journal of Biological Sciences 23:1207-1220-   Cullen et al. (1987) Sequence and centromere proximal location of a    transformation enhancing fragment ans1 from Aspergillus nidulans.    Nucleic Acids Research 15: 9163-75-   J. Dassa et al. (1990) The complete nucleotide sequence of the    Escherichia coli gene appA reveals significant homology between pH    2.5 acid phosphatase and glucose-1-phosphatase. J. Bacteriol.    172:5497-5500-   Gems et al. (1991) An autonomously replicating plasmid transforms    Aspergillus nidulans at high frequency. Gene 98:61-67-   Greiner et al, Purification and characterization of two phytases    from E. Coli. Arch. Biochem. Biophys., 303, 107-113, 1993-   J. C. Janson and L. Ryden (1989) Protein Purification, VCH    Publishers, New York-   T. Karhunen, A. Mäntylä, K. M. H. Nevalainen, P. L. Suominen (1993)    High frequency one-step gene replacement in Trichoderma reesei. I.    Endoglucanase I overproduction. Mol Gen Genet 241: 515-522-   U.K. Laemmli (1970) Cleavage of structural protein during the    assembly of the head of bacteriophage T4. Nature 227: 680-685-   Lim et al., 2000, Crystal structures of Escherichia coli phytase and    its complex with phytate. Nat. Struct. Biol. 7: 108-113-   Needleman and Wunsch (1970) A general method applicable to the    search for similarities in the amino acid sequence of two    proteins. J. Mol. Biol. 48: 443-   Oshima et al. (1996) A 718-kb DNA sequence of the Escherichia coli    K-12 genome corresponding to the 12.7-28.0 min region on the linkage    map. DNA Research, 3:137-155-   Pearson and Lipman (1988) Enhanced tools for biological sequence    comparison. Proc. Natl. Acad. Sci. USA 85 (8): 2444-8-   M. Penttilä, H. Nevelainen, M. Rättö, E. Salminen, J. Knowles (1987)    A versatile transformation system for the cellulolytic filamentous    fungus Trichoderma reesei. Gene 61: 155-164-   V. K. Powar and V. Jagannathan (1982) Purification and properties of    phytate-specific phosphatase from Bacillus subtilis. Journal of    Bacteriology 151:1102-1108-   U. Raeder, P. Broda (1985) Rapid preparation of DNA from filamentous    fungi. Lett Appl Microbiol 1: 17-20-   T. J. Richmond (1984) Solvent accessible surface area and excluded    volume in proteins. J. Mol. Biol., 178, 63-89-   Rodriguez et al. (2000) Site-directed mutagenesis improves catalytic    efficiency and thermostability of Escherichia coli pH 2.5 acid    phosphatase/phytase expressed in Pichia pastoris. Arch. Biochem.    Biophys., 382:105-112-   Saboulard et al (2005) High-throughput site-directed mutagenesis    using oligonucleotides synthesized on DNA chips. Biotechniques Sep    39(3): 363-8)-   Smith and Waterman (1981) Ad. App. Math. 2: 482-   Touati and Danchin (1987) The structure of the promoter and amino    terminal region of the pH 2.5 acid phosphatase structural gene    (appA) of E. coli: a negative control of transcription mediated by    cyclic AMP. Biochimie, 69:215-221-   Wyss et al (1999) Biochemical Characterization of Fungal Phytases.    Appl Environ Microbiol 65 (2) 367-373

TABLE 1 P3L P3V V4G A5P P8N P8V T9I T9Q T9S T9Y G10A G10P V16M V17W L19GS20C R21F H22A H225 H22Y G23S R25C P27F T28N T28S T28V Q30R Q32R L33RT41G G50D G50E V60I Y67F Y67W G75R A78P D92R D94G D945 Q95V R96A T97NV125N D126Q H130N H130Q H130R H130W P131S V132W D133G D133P D133R D133VD133W D140E D140F L152N L152P L156N F167N C182N F197V K206A T209C K210CK210E K210S K210T K210V K210Y V211C V211G L217N L223S E225D F227S L229HQ230T N231K A234K P236N S251N L252M H256A H256E H256P N257I Q259K Q259YD261F A264P Y268N Y268E K273N G274S P276L Q292P P297S Q301L H310R D311AD311E D311G T312D T312N T312P T312V N313F N313R I314E I314M A323E P331RD332A D332L D332Q N333V P335C P335G P335R P335S V341A V341E E343A E343GD349N R353C Y354N V355W A370D A370T K376S P379L G381S 5393G P414Q

TABLE 2 <35% 35% < × ASA <35% ASA <35% ASA <70% ASA >70% ASA Q7N S220NP335S Y51N K29N D36N Q230N P335T C81N T142N P39N Q233S Q352S Q95N A177NR98N Q233T Q352T D140N A177S Q143N + R242N E371S P155N A177T H145T E158NQ259S E371T G189N K201N E158N + Q259T P379S K210N G293N S160T P207NA264N P379T I250S Q326S P207S K273N A380S I250T Q326T P207T P300N A380TP297N E391N 5212N H310N G394S D349S A218N D332N G394T D349T

TABLE 3 SER20-GLY339 VAL24-GLY56 SER26-TRP45 GLN30-MET34 LEU52-LEU99THR53-GLY56 ALA57-ALA103 GLY101-VAL116 ALA147-TYR268 ASP193-ASN196GLY274-ASN316 GLY322-ASP388

TABLE 4 Table 4A Mutants K210S Y268E Q292P 80%-20% activity 1.75 1.982.3 extinction coefficients

Table 4B Wild type K210S T, ° C. 80% 50.77 50.51 T, ° C. 20% 53.44 55.17Delta. 80%-20% 2.67 4.66 R (K210S/WT) 1.74531835

Table 4C Mutants T142N A177T Q326T 80%-20% activity 1.62 1.62 1.52extinction coefficients

Table 4D Mutants G274C/N316C(SS11) 80%-20% activity 2.33 extinctioncoefficients

TABLE 5 Table 5A L19 S20 R21 H22 G23 V24 R25 S26 P27 T28 K29 Q30 T31 M34A49 G50 Y51 L52 D92 V93 D94 Q95 R96 T97 R98 T100 V125 D126 F129 H130D133 P207 T209 K210 V211 S212 L213 L217, A218, L219, S220, S221, T222L223 G224 E225 I226 F227 L228 L229 L253 L255 H256 N257 Q259 F260 M263A264 K273 L277 G308 G309 H310 D311 T312 N313 I314 A315 N316 D332 N333T334 P335 P336 G337 G338

Table 5B Q30D Y51G Y51Q Y51W L52G V93G R98T F129W H130Y P155T L219VL255T M263L G308S

1. An enhanced phytase variant whose sequence is SEQ ID NO: 1 or afunctional derivative thereof, comprising at least one substitution onone of the amino acids from the group consisting of P3, V4, A5, P8, T9,G10, V16, V17, L19, S20, R21, H22, G23, V24, R25, S26, P27, T28, K29,Q30, T31, Q32, L33, M34, D36, P39, K41, W45, A49, G50, Y51, L52, T53,G56, A57, V60, Y67, G75, A78, C81, D92, V93, D94, Q95, R96, T97, R98,L99, T100, G101, A103, V116, V125, D126, F129, H130, P131, V132, D133,D140, T142, Q143+H145, A147, L152, P155, L156, E158, E158+S160, F167,A177, C182, G189, D193, N196, F197, K201, K206, P207, T209, K210, V211,S212, L213, L217, A218, L219, S220, S221, T222, L223, G224, E225, I226,F227, L228, L229, Q230, N231, Q233, A234, P236, R242, I250, S251, L252,L253, L255, H256, N257, Q259, F260, D261, M263, A264, Y268, K273, G274,P276, L277, Q292, G293, P297, P300, Q301, G308, G309, H310, D311, T312,N313, I314, A315, N316, G322, A323, Q326, P331, D332, N333, T334, P335,P336, G337, G338, G339, V341, E343, D349, Q352, R353, Y354, I355, A370,E371, K376, P379, A380, G381, D388, E391, S393, G394 and P414, thepositions being indicated in SEQ ID NO:
 1. 2. The enhanced phytasevariant whose sequence is SEQ ID NO: 1 or a functional derivativethereof according to claim 1, comprising at least one substitutionselected from the group consisting of P3L, P3V, V4G, ASP, P8N, P8V, T9I,T9Q, T9S, T9Y, G10A, G10P, V16M, V17W, L19G, 520C, R21F, H22A, H22S,H22Y, G23S, V24C, R25C, S26C, P27F, T28N, T28S, T28V, K29N, Q30C, Q30D,Q30R, Q32R, L33R, M34C, D36N, P39N, K41G, W45C, G50D, G50E, Y51G, Y51N,Y51Q, Y51W, L52C, L52G, T53C, G56C, A57C, V60I, Y67F, Y67W, G75R, A78P,C81N, D92R, V93G, D94G, D94S, Q95N, Q95V, R96A, T97N, R98N, R98T, L99C,G101C, A103C, V116C, V125N, D126Q, F129W, H130N, H130Q, H130R, H130W,H130Y, P131S, V132W, D133G, D133P, D133R, D133V, D133W, D140E, D140F,D140N, T142N, Q143N+H145T, A147C, L152N, L152P, P155N, P155T, L156N,E158N, E158N+5160T, F167N, A177N, A177S, A177T, C182N, G189N, D193C,N196C, F197V, K201N, K206A, P207N, P207S, P207T, T209C, K210C, K210E,K210N, K210S, K210T, K210V, K210Y, V211C, V211G, S212N, L217N, A218N,L219V, S220N, L223S, E225D, F227S, L229H, Q230N, Q230T, N231K, Q233S,Q233T, A234K, P236N, R242N, I250S, I250T, S251N, L252M, L255T, H256A,H256E, H256P, N257I, Q259K, Q259S, Q259T, Q259Y, D261F, M263L, A264N,A264P, Y268C, Y268E, Y268N, K273N, G274C, G274S, P276L, Q292P, G293N,P297N, P297S, P300N, Q301L, G308S, H310N, H310R, D311A, D311E, D311G,T312D, T312N, T312P, T312V, N313F, N313R, I314E, I314M, N316C, G322C,A323E, Q326S, Q326T, P331R, D332A, D332L, D332N, D332Q, N333V, P335C,P335G, P335R, P335S, P335T, G339C, V341A, V341E, E343A, E343G, D349N,D349S, D349T, Q352S, Q352T, R353C, Y354N, I355W, A370D, A370T, E371S,E371T, K376S, P379L, P379S, P379T, A380S, A380T, G381S, D388C, E391N,S393G, G394S, G394T and P414Q, the positions being indicated in SEQ IDNO:
 1. 3. The enhanced phytase variant whose sequence is SEQ ID NO: 1 ora functional derivative thereof according to claim 2, comprising atleast one substitution selected from the group consisting of P8N, P8V,T9I, T9S, G10A, G10P, V16M, V17W, S20C, G23S, V24C, S26C, P27F, T28N,T28S, K29N, Q30C, Q30D, Q30R, Q32R, L33R, M34C, D36N, P39N, K41G, W45C,G50D, G50E, Y51G, Y51N, Y51Q, Y51W, L52C, L52G, T53C, G56C, A57C, V60I,Y67F, G75R, A78P, C81N, V93G, Q95N, T97N, R98N, R98T, L99C, G101C,A103C, V116C, F129W, H130N, H130Q, H130R, H130Y, P131S, V132W, D133G,D140F, D140N, T142N, Q143N+H145T, A147C, L152N, L152P, P155N, P155T,L156N, E158N, E158N+S160T, F167N, A177N, A177S, A177S+Q326S,A177S+Q326T, A177T, C182N, G189N, D193C, N196C, F197V, K201N, K206A,P207N, P207S, P207T, K210N, K210S, K210T, K210Y, V211G, S212N, L217N,A218N, L219V, S220N, L223S, L229H, Q230N, N231K, Q233S, Q233T, A234K,P236N, R242N, I250S, I250T, S251N, L252M, L255T, H256E, N257I, Q259S,Q259T, M263L, A264N, A264P, Y268C, Y268E, Y268N, G274C, Q292P, G293N,P297N, P297S, P300N, Q301L, G308S, H310N, T312D, T312N, I314E, I314M,N316C, G322C, A323E, Q326S, Q326T, P331R, D332A, D332L, D332N, D332Q,P335C, P335S, P335T, G339C, V341E, E343A, E343G, D349N, D349S, D349T,Q352S, Q352T, A370D, E371S, E371T, K376S, P379L, P379S, P379T, A380S,A380T, G381S, D388C, E391N, S393G, G394S, G394T and P414Q, the positionsbeing indicated in SEQ ID NO:1.
 4. The enhanced phytase variant whosesequence is SEQ ID NO: 1 or a functional derivative thereof according toclaim 1, comprising at least one substitution on one of the amino acidsfrom the group consisting of K29, Q30, Y51, L52, G75, C81, V93, Q95,R98, L99, F129, H130, D140, T142, P155, F167, A177, G189, K201, K210,L219, I250, S251, L252, L255, M263, Y268, G274, Q292, G293, P297, G308,N316, Q326, D349 and E391, the positions being indicated in SEQ IDNO:
 1. 5. The enhanced phytase variant sequence is SEQ ID NO: 1 or afunctional derivative thereof according to claim 4, wherein thesubstitutions on the amino acids K29, Q30, Y51, L52, G75, C81, V93, Q95,R98, L99, F129, H130, D140, D140N, T142, P155, F167, A177, G189, K201,K210, L219, I250, S251, L252, L255, M263, Y268, G274, Q292, G293, P297,G308, N316, Q326, D349 and E391 are selected from the group consistingof K29N, Q30D, Y51G, Y51N, Y51Q, Y51W, L52C, L52G, G75R, C81N, V93G,Q95N, R98T, L99C, F129W, H130Y, D140F, D140N, T142N, P155N, P155T,F167N, A177N, A177S, A177T, G189N, K201N, K210N, K210S, L219V, I250S,I250T, S251N, L252M, L255T, M263L, Y268E, Y268N, G274C, Q292P, G293N,P297N, G308S, N316C, Q326S, Q326T, D349S, D349T and E391N, the positionsbeing indicated in SEQ ID NO:
 1. 6. The enhanced phytase variant whosesequence is SEQ ID NO: 1 or a functional derivative thereof according toclaim 1, comprising a combination of substitutions selected from thegroup consisting of G274C+N316C, T142N+A177T+Q326T, K210S+Y268E+Q292P,D140F+Y268E+Q292P, F167N+Y268E+Q292P, T142N+A177T+K210S+Q326T,T142N+A177T+K210S+Y268E+Q292P+Q326T,T142N+A177T+K210S+Y268E+Q292P+Q326T+G274C+N316C, andL52C+L99C+T142N+A177T+K210S+Y268E+Q292P+Q326T, the positions beingindicated in SEQ ID NO:
 1. 7. The enhanced phytase variant whosesequence is SEQ ID NO: 1 or a functional derivative thereof according toclaim 1, comprising a combination of substitutions consisting ofT142N+A177T+K210S+Y268E+G274C+Q292P+N316C+Q326T, the positions beingindicated in SEQ ID NO:
 1. 8. The enhanced phytase variant whosesequence is SEQ ID NO: 1 or a functional derivative thereof according toclaim 1, comprising a combination of substitutions consisting ofT142N+A177T+K210S+Y268E+Q292P+Q326T, the positions being indicated inSEQ ID NO:
 1. 9. The enhanced phytase variant whose sequence is SEQ IDNO: 1 or a functional derivative thereof according to claim 1, whereinits sequence is SEQ ID NO: 1 with the selected substitution orsubstitutions.
 10. A nucleic acid coding for the enhanced phytasevariant according to claim
 1. 11. A cassette or expression vector forthe nucleic acid according to claim
 10. 12. A host cell comprising thenucleic acid according to claim 10 or an expression vector thereof. 13.A composition comprising at least one enhanced phytase variant withsequence SEQ ID NO: 1 or a functional derivative thereof according toclaim 1 with the selected substitution or substitutions.
 14. A methodfor preparing a food additive or an animal feed comprising a use of anenhanced phytase variant whose sequence is SEQ ID NO: 1 or a functionalderivative thereof according to claim 1 with the selected substitutionor substitutions.
 15. A method for producing an enhanced phytase variantcomprising a use of a nucleic acid according to claim 10, or anexpression cassette thereof, or a cell comprising said nucleic acid. 16.An animal feed comprising an enhanced phytase variant whose sequence isSEQ ID NO:1 or a functional derivative thereof according to claim 1 withthe selected substitution or substitutions.